An antibody, GC-17, thoroughly characterized for its specificity for estrogen receptor-beta (ER-beta), was used to immunolocalize the receptor in histologically normal prostate, prostatic intraepithelial neoplasia, primary carcinomas, and in metastases to lymph nodes and bone. Comparisons were made between ER-beta, estrogen receptor-alpha (ER-alpha), and androgen receptor (AR) immunostaining in these tissues. Concurrently, transcript expression of the three steroid hormone receptors was studied by reverse transcriptase-polymerase chain reaction analysis on laser capture-microdissected samples of normal prostatic acini, dysplasias, and carcinomas. In Western blot analyses, GC-17 selectively identified a 63-kd protein expressed in normal and malignant prostatic epithelial cells as well as in normal testicular and prostatic tissues. This protein likely represents a posttranslationally modified form of the long-form ER-beta, which has a predicted size of 59 kd based on polypeptide length. In normal prostate, ER-beta immunostaining was predominately localized in the nuclei of basal cells and to a lesser extent stromal cells. ER-alpha staining was only present in stromal cell nuclei. AR immunostaining was variable in basal cells but strongly expressed in nuclei of secretory and stromal cells. Overall, prostatic carcinogenesis was characterized by a loss of ER-beta expression at the protein and transcript levels in high-grade dysplasias, its reappearance in grade 3 cancers, and its diminution/absence in grade 4/5 neoplasms. In contrast, AR was strongly expressed in all grades of dysplasia and carcinoma. Because ER-beta is thought to function as an inhibitor of prostatic growth, androgen action, presumably mediated by functional AR and unopposed by the beta receptor, may have provided a strong stimulus for aberrant cell growth. With the exception of a small subset of dysplasias in the central zone and a few carcinomas, ER-alpha-stained cells were not found in these lesions. The majority of bone and lymph node metastases contained cells that were immunostained for ER-beta. Expression of ER-beta in metastases may have been influenced by the local microenvironment in these tissues. In contrast, ER-alpha-stained cells were absent in bone metastases and rare in lymph nodes metastases. Irrespective of the site, AR-positive cells were found in all metastases. Based on our recent finding of anti-estrogen/ER-beta-mediated growth inhibition of prostate cancer cells in vitro, the presence of ER-beta in metastatic cells may have important implications for the treatment of late-stage disease.

G protein–coupled receptor 30 (GPR30) exhibits estrogen-binding affinity and mediates nongenomic signaling of estrogen to regulate cell growth. We here demonstrated for the first time, in contrast to the reported promoting action of GPR30 on the growth of breast and ovarian cancer cells, that activation of GPR30 by the receptor-specific, non-estrogenic ligand G-1 inhibited growth of androgen-dependent and -independent prostate cancer (PCa) cells in vitro and PC-3 xenografts in vivo. However, G-1 elicited no growth or histological changes in the prostates of intact mice and did not inhibit growth in quiescent BPH-1, an immortalized benign prostatic epithelial cell line. Treatment of PC-3 cells with G-1-induced cell-cycle arrest at the G2 phase and reduced the expression of G2-checkpoint regulators (cyclin A2, cyclin B1, cdc25c, and cdc2) and the phosphorylation of their common transcriptional regulator NF-YA in PC-3 cells. With the extensive use of siRNA knockdown experiments and the MEK inhibitor PD98059 in the present study, we dissected the mechanism underlying G-1–induced inhibition of PC-3 cell growth, which was mediated through GPR30, followed by a sustained activation of Erk1/2 and a c-jun/c-fos-dependent upregulation of p21, resulting in the arrest of PC-3 growth at the G2 phase. The discovery of this signaling pathway lays the foundation for future development of GPR30-based therapies for PCa.

Our understanding of the roles played by sex hormones in ovarian carcinogenesis has been limited by a lack of data concerning the mode of sex hormone action in human ovarian surface epithelial (HOSE) cells, the tissue of origin of >90% of ovarian cancers. We have compared the relative abundance of estrogen receptor (ER)␣, ER␤, progesterone receptor (PR), and androgen receptor (AR) mRNA in four primary cultures of HOSE cells obtained from postmenopausal women to those found in late serous adenocarcinoma primary cell cultures and established ovarian cancer cell lines. We observed coexpression of ER␣ and ER␤ mRNA along with AR and PR transcripts in normal HOSE cells and disruption of ER␣ mRNA expression as well as dramatic down-regulation of PR and AR transcript expression in most ovarian cancer cells. In contrast, levels of ER␤ mRNA were unaffected by the malignant state. Additionally, a novel mutation involving a 32-bp deletion in exon 1 of ER␣ transcripts was detected in the SKOV3 cell line. This mutation would explain why SKOV3 was reported to be ER-positive but estrogen-insensitive. Taken together, these findings suggest that estrogens, signaling via either or both ER subtypes, may play an indispensable role in regulating normal HOSE cell functions. Therefore, loss of ER␣, PR, and AR mRNA expression in HOSE cells may be responsible for neoplastic transformation in this cell type. In contrast, the roles played by ER␤ in normal and malignant HOSE cells remain elusive. Finally, the coexistence of mutated ER␣ mRNA and normal ER␤ transcripts in SKOV3 argues in favor of a dependency of ER␤ action on functional ER␣s.